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Characterization of endosomes and interactions from TMEM230 and TMEM9/9B mutants in human neurons

ABSTRACT: EndoMAP aims to systematically dissect the human endosomal protein complexes. To examine TMEM9 and TMEM230 interactions, we generated TMEM230 and TMEM9 knockouts (KOs) using CRISPR/Cas9 in human embryonic stem (ES) cells and converted the cells to induced neurons (iNeurons). TMEM230 and TMEM9 were immunoprecipitated (IP) on day-21 iNeurons and analyzed by TMT-MS, using the respective KOs as a negative control. In addition, TMEM230 immunoprecipitations were performed in TMEM230 KO iNeurons expressing WT and individual disease variant of TMEM230 (Y29C, R78L, and X121W), as well as a triple mutant carrying all three variants. To examine the impact of TMEM230 mutations and TMEM9/9B loss on total and endosomal proteome, we additionally generated TMEM230(X121W) and TMEM9/TMEM9B double knockout (DKO) in ES cells. The proteome of post-nuclear supernatant (PNS) and purified endosomes (Endo-IP) from WT, TMEM230 KO, and TMEM230(X121W) iNeurons were analyzed by TMT-MS. The proteome of PNS and purified endosomes (Endo-IP) from TMEM9 KO and two clones of TMEM9/9B DKO iNeurons were analyzed by TMT-MS.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Miguel A. Gonzalez-Lozano

LAB HEAD: J. Wade Harper

PROVIDER: PXD054765 | Pride | 2025-03-29

REPOSITORIES: Pride

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			Action	DRS
	618076_ea16542.mzid.gz	Mzid
	618077_ea16541.mzid.gz	Mzid
	618207_ea16538.mzid.gz	Mzid
	618208_ea16539.mzid.gz	Mzid
	623123_ea17034.mzid.gz	Mzid

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